Hypoxia enhances autophagy level of human sperms

The relationship between oxygen sensing and autophagy in human sperms was explored in this study. Health semen and asthenozoospermia (astheno) semen were incubated with hypoxia-inducible factor-1α (HIF-1α) interferents, i.e., lificiguat (YC-1) or cobalt chloride (CoCl2), respectively. Label-free quantitative proteomic technology was used to identify the differentially expressed proteins in human semen under the hypoxia condition. Selected proteins were detected with ELISA. It was found that the autophagy levels of sperm in the YC-1 + health group or CoCl2 + astheno group increased while the vitality decreased. A total of 17, 34 and 35 differentially expressed proteins were observed in the Astheno group, the YC-1 + health group and the CoCl2 + astheno group, respectively. These proteins were primarily associated with protein processing in endoplasmic reticulum, Th17 cell differentiation, progesterone-mediated oocyte maturation, glycolysis/gluconeogenesis, HIF-1 signaling pathway, biosynthesis of amino acids, and carbon metabolism. The expression levels of protein HIF-1α, LC3B, histone H4, cathepsin L and ENO1 changed significantly in the groups. The study suggests that hypoxia can increase sperm autophagy level and reduce their vitality through HIF-1 signaling pathway and glycolysis/gluconeogenesis signaling pathway. Furthermore, proteins histone H4, cathepsin L, glutathione synthetase and ENO1 are proposed as potential biomarkers of autophagy and vitality in asthenozoospermia sperm.


Study population
The study participants were randomly selected form individuals who visited the Centre of Reproductive Medicine, Affiliated Hospital of Guilin Medical University, between June 2021 and December 2022.The participants consisted of individuals with health semen quality and patients diagnosed with astheno.To ensure comparability, the two categories were matched based on age frequency.
Both the health semen samples and the astheno semen samples met the following criteria: (1) Semen volume greater than 1.5 mL; (2) Semen pH value ranging from 7.2 to 7.8; (3) Sperm concentration equal to or higher than 15 × 10 6 cell/mL; (4) Liquefaction time of no more than 30 min; (5) Color of the semen: gray-white or light yellow; (6) Total sperm motility, which includes progressive (PR) and non-progressive (NP) motility, of the health semen had to be greater than 40%, with PR motility equal to or greater than 32%.In contrast, the astheno group had PR motility less than 32%.The inclusion criteria for the study were as the followings: (1) Males between the ages of 18 and 45; (2) No history of severe or significant environmental exposures such as heavy smoking, exposure to chemicals, or pesticides.The exclusion criteria were as the followings: (1) Individuals with a history of severe reproductive system trauma; (2) Long-term use of steroid drugs or medicines that affected sperm function; (3) Individuals with severe necrospermia, azoospermia, or cryptorchidism.
A questionnaire was used on-site to collect general and clinical data of the subjects, including name, age, smoking, drinking (alcohol consumption), medication history, sampling time, and abstinence days.

Semen sample collection and treatment
The semen samples were collected through masturbation after abstinence of 3-7 days.The samples were kept warm at a temperature between 20 and 37 ℃ and were tested within 1 h.Semen is a mixture of sperm and secretions from the male reproductive organs.Sperm accounts for about 5-10% of the total ejaculate volume, while the remaining 90-95% (referred to as seminal plasma) primarily consists of water, proteins, polypeptides, carbohydrates, enzymes, inorganic salts, and organic small molecules.These components play crucial roles in sperm function and offer potential biomarkers for assessing male fertility.The testicular barrier effectively isolates and maintain the stability and specificity of seminal plasma components.Therefore, complete semen samples, which include both seminal plasma and sperm, were used for semen quality analysis and proteomics analysis in this study.
Soluble guanylate cyclase (sGC) activator (lificiguat, YC-1, Selleckchem, USA) has been shown to completely suppress the expression of HIF-1α under hypoxia condition 20 .On the other hand, Cobalt chloride (CoCl 2 , Mackin, China), a chemical hypoxia mimetic, can create a hypoxic environment and induce the accumulation of HIF-1α, inducing hypoxic damage in various cell models in vitro 21 .In this study, YC-1 and CoCl 2 were used to incubate human semen samples to explore the effects of HIF-1α on autophagy and elucidate the underlying mechanisms.
A total of 85 qualified semen samples were included in the study and divided into 4 groups.The 41 samples of health semen were further randomly categorized into a health control group (Health, 20 samples) and a health experimental group (YC-1 + health, 21 samples).Similarly, the 44 samples of asthenozoospermia semen were divided into astheno control group (Astheno, 22 samples) and astheno experimental group (CoCl 2 + astheno, 22 samples).According to the results of our pilot study, the semen samples of YC-1 + health group or that of the CoCl 2 + astheno group were incubated with 5000 μM YC-1 or 100 nM CoCl 2 at 37 ℃ for 60 min, respectively.Health group and Astheno group were not given any of the reagents.

Sperm motility assay
The semen was gently and thoroughly mixed, and then 0.1 mL of the sample was applied on a slide.A computeraided sperm analysis system (CASA, ZJ-3000E, Xuzhou, China) was used to analyze sperm concentration and PR (%).

Semen sample purification
The SWIM-UP method was employed to purify the semen samples.Briefly, a total of 2 mL preheated fallopian tube fluid (HTF, MCE, USA) at 37 ℃ human and 500 μl semen sample were separately added to a centrifuge tube.A clear boundary line between the semen and HTF solution was created so as to segregate the sperms based on their motility.The centrifuge tube was tilted at 45° angle and incubated at 37 ℃ for 1 h.

Experimental procedures of MDC method
Following the instructions of the MDC (Monodansylcadaverin) autophagy staining test kit (Solarbio, China), 875 μl of the supernatant was collected and centrifuged at 700g for 5 min.The cells were dispersed with 1 × Wash buffer and centrifuged at 800 g for 5 min to collect the cells.The cells were re-suspended in 1 × Wash buffer, counted, and adjusted to a concentration of 1 × 10 6 cell/mL.A volume of 90 μl of the cell suspension was added into an EP tube and stained with 10 μl of MDC for 30 min in darkness.The cells were then washed with 400 μl of 1 × Wash buffer and centrifuged.This washing process was repeated twice.The cells were finally re-suspended in 100 μl of collection buffer.A volume of 10 μl of the cell suspension was plated on a glass slide, covered, and

Protein extraction and digestion
The semen sample was combined with lysis buffer (Promega, US) and sonicated for 5 min in an ice bath.The lysis buffer consisted of 8M urea (GibcoBRL, US), 30 mM HEPES (AMRECSO, US), 1 mM PMSF (AMRECSO, US), 2 mM EDTA (AMRECSO, US) and 10 mM DTT (Promega, US).Subsequently, the samples were centrifuged at 20,000g for 30 min.The supernatant, along with 10 mM DTT, was incubated at 56 ℃ for 1 h and then mixed with 55 mM IAM (Promega, US), followed by incubation in darkness for 1 h.After centrifugation at 20,000g and 4 ℃ for 30 min, the protein concentration was determined using the Bradford method.
Once protein extraction was completed, digestion was performed.40 μg of protein from each sample was transferred to a 3K ultrafiltration tube and centrifuged at 14,000g and 4 ℃ for 40 min.Then, 200 μl of 50 mM NH 4 HCO 3 was added to the sediments and centrifuged again.This process repeated twice.Next, 1 μg/μL trypsin was added to the sample at a protein substrate to enzyme ratio of 30:1, and the mixture was incubated in a water bath at 37 ℃ for 24 h.After the digestion solution was lyophilized, the peptide segments were re-suspended in 30μL 25 mM NH 4 HCO 3 .
After mass spectrometry scanning, the mass spectrometry raw file was obtained.MaxQuant software (https:// www.maxqda.com/, version: 1.6.0.1) was applied for data input, screening and quantitative analysis of the raw files.The screening parameters, retrieval and quantitative parameters are detailed below.
Quantitative parameters: Protein ratio type and Normalisation method: Intensity; Minimum peptides: 1.The significance of protein differences was evaluated using MSstats variance analysis in R software (http:// www.rrsoft ware.com/, version 4.1.0).The criteria for screening differentially expressed proteins were as follows: (1) The fold change in protein average abundance was greater than 1.2 or less than 0.83, and (2) The p-value of T-test was less than 0.05.

Detection of related protein using ELISA
The semen sample was adjusted to a cellular concentration of 1 × 10 6 cell/mL with PBS (pH 7.2-7.4).After undergoing several cycles of freezing and thawing, 0.5 mL of lysis solution was added to lyse the cells on ice.The mixture was then centrifuged to collect the proteins, and the concentration of total protein was measured with Bradford method.
The levels of glutathione synthetase in the semen samples were determined using the Human Glutathione Synthetase ELISA Kit (Ruichuang, Shanghai, China).Following the kit's instructions, varied concentrations of standard samples (50 μL each) were added into the standard wells.Blank wells (without samples and enzyme conjugate) and test sample wells were also prepared.In the test sample wells on the coated plate, 40 μL of sample diluent was added first, followed by the addition of 10 μL of the test sample (resulting in a final dilution factor of 5).The samples were placed at the bottom of the plate wells, ensuring no contact with the well walls, and gently mixed.Enzyme conjugate (100 μL) was added to each well (except the blank ones).The plate was sealed with a sealing film and incubated at 37°C for 60 min.After carefully removing the sealing film, the liquid was discarded, and the plate was tapped to remove water droplets.Each well was filled with washing buffer, and then dried.Next, 50 μL of reagent A and 50 μL of reagent B were added in each well.The plate was gently shaken to mix the contents and incubated at 37 °C in darkness for 15 min for color development.Subsequently, 50 μL of stop solution was added to terminate the reaction (turning the color from blue to yellow).The absorbance (OD value) was measured at a wavelength of 450 nm, with the blank well used for zero calibration.The measurement was performed within 15 min after adding the stop solution.The concentrations of the samples were calculated using the linear regression curve equation derived from the standard samples.

Effect of hypoxia on sperm autophagy level
Compared with the Health control group, the degree of sperm autophagy was significantly increased after 60 min of treatment with 5000 µM YC-1.Similarly, compared with the Astheno control group, the sperm autophagy rate was increased after 60 min of treatment with 100 nM CoCl 2 (Fig. 2).

Proteomics results
Protein and peptide content determination A total of 12 label-free quantitative proteomics experiments were conducted on 3 samples from each of the 4 groups.The protein concentrations of all 4 groups were determined to be 1.50 ± 0.14 μg/μL, indicating a reasonable range of protein concentrations.SDS-PAGE gel electrophoresis analysis was performed on each group of protein samples.Every protein bands in the electrophoresis plates were clear and not obviously degraded, suggesting that the samples were suitable for further analysis.

Differential protein screening
Distinct molecular peaks were observed in the total ion chromatography, and the main fragment ion peaks were found to be relevant to the protein quality.
In comparison to the Health group, the Astheno group showed 17 differentially expressed proteins, including 5 up-regulated and 12 down-regulated proteins.The YC-1 + health group exhibited 34 differentially expressed proteins compared to the Health group, with 1 up-regulated and 33 down-regulated proteins.Similarly, when compared with Astheno group, the CoCl 2 + astheno group revealed 35 differentially expressed proteins, all which were down-regulated (Table 2).

Clustering analysis
The clustering heat map clearly indicated that the separation of differentially expressed proteins between the Astheno group and Health group, YC-1 + health group and Health group, as well as the CoCl 2 + astheno group and Astheno group.This suggests the identified proteins represented significant inter-group differences of the semen samples (Fig. 3).www.nature.com/scientificreports/Gene ontology (GO) analysis GO classification annotation (refer to Appendix 1 Fig. 1) revealed that the differentially expressed semen proteins in the Astheno group were primarily associated with IgA immunoglobulin complex, ubiquitin ligase complex, endoplasmic reticulum quality control compartment, and more.The molecular functions basically were binding and enzyme activity.The biological processes were mainly related to protein transport, protein localization, protein targeting and respiratory burst (refer to Appendix 1 Fig. 1A).www.nature.com/scientificreports/ The differential proteins in the YC-1 + health group were mainly distributed in the endoplasmic reticulum exit site, presynaptic active zone, phosphopyruvate hydratase complex, bicellular tight junction and chloride channel complex.The molecular functions primarily included NADP binding and enzyme activity.The biological processes were involved in biosynthetic process and metabolic process (refer to Appendix 1 Fig. 1B).
The differential proteins in the CoCl 2 + astheno group semen were primarily found in the endoplasmic reticulum lumen, extracellular matrix, lysosome lumen, vacuolar membrane and cytoplasmic vesicle lumen.The molecular function included protein binding, enzyme activity and extracellular matrix structural constituent.The biological process was associated with metabolic process, negative regulation of enzyme activity, glycolytic process and positive regulation of oxidative stress-induced cell death (refer to Appendix 1 Fig. 1C).
KEGG pathway enrichment analysis (www.kegg.jp/ kegg/ kegg1.html) (refer to Appendix 2 Table 1).The differentially expressed human semen proteins in the Astheno group were found to be involved in various metabolic pathways, including protein processing in endoplasmic reticulum, Th17 cell differentiation, progesterone-mediated oocyte maturation, and more.
The differential proteins in the YC-1 + health group primarily participated in biosynthesis of amino acids, metabolism, glycolysis/gluconeogenesis, HIF-1 signaling pathway, and other pathways.
The differential proteins in the CoCl 2 + astheno group mainly contributed to glycolysis/gluconeogenesis, biosynthesis of amino acids and fatty acid, carbon metabolism, complement and coagulation cascades, HIF-1 signaling pathway, PI3K-Akt signaling pathway, and more.

Protein-protein interaction (PPI) network analysis (https:// www. unipr ot. org)
According to STRING analysis, the PPI of differential proteins in the YC-1 + health group (Fig. 4A) was primarily associated with glycolysis/gluconeogenesis, biosynthesis of amino acids, carbon metabolism, HIF-1 signaling pathway and others.The proteins were mainly expressed in semen and lymph.
The PPI in the CoCl 2 + astheno group (Fig. 4B) was mainly involved in the biological processes such as positive regulation of protein insertion into the mitochondrial membrane, apoptotic signaling pathways, canonical glycolysis, regulation of plasminogen activation, gluconeogenesis, negative regulation of coagulation and more.The proteins were associated with pathways including glycolysis/gluconeogenesis, biosynthesis of amino acids, HIF-1 signaling pathway and PI3K/AKT signaling pathway.
The PPIs observed were consistent with GO and KEGG pathway annotations.

Joint analysis of differential proteins between groups
The intersection proteins of differentially expressed proteins in the YC-1 + health group, CoCl 2 + astheno group, and Astheno group were integrated to identify the up-regulated or down-regulated trends after reagent interference.Compared with the Health group, the Q0VAS5 protein (Histone H4, His H4) was up-regulated in the YC-1 + health group, while A0A7I2V5M3 protein (Cathepsin L, Cath L), E9PKT6 protein (Cathepsin H), P58499 protein (Protein FAM3B), Q53YD7 protein (Elongation factor 1-gamma) were down-regulated.These differences resulted in an increased level of sperm autophagy and decreased sperm motility.Furthermore, in comparison with the Health group, the A0A2R8Y430 protein (Glutathione synthetase, Glu), Q9UM22 protein (Mammalian ependymin-related protein 1), Q9Y6R7 protein (IgGFc-binding protein), P58499 protein (Protein FAM3B), Q13438 protein (Protein OS-9) were down-regulated in the CoCl 2 + astheno group.This suggests that CoCl 2 inhibited the expression of these proteins, leading to an and enhanced level of sperm autophagy and reduction in sperm motility.www.nature.com/scientificreports/

Expression levels of selected proteins in human semen
The expression levels of related proteins are presented in Table 3. HIF-1αand LC3B exhibited high expression in both the YC-1 + health group and the CoCl 2 + astheno group.Compared with the Health group, the level of glycolysis-related protein enolase 1 (ENO1) did not change significantly in the YC-1 + health group.However, compared to the Astheno group, the ENO1 level was higher in the CoCl 2 + astheno group.The levels of the glycolysis-related protein PGK1 did not differ between the Health group and the YC-1 + health group, as well as between the Astheno group and the CoCl 2 + astheno group.Additionally, in comparison to the Health group, the A0A2R8Y430 protein (Glutathione synthetase) was down-regulated in the CoCl 2 + astheno group, the A0A7I2V5M3 protein (Cathepsin L) was down-regulated, and the Q0VAS5 protein (Histone H4) was highly expressed in the YC-1 + health group.The levels of HIF-1α, LC3B, ENO1, and PGK1 in the Astheno group differed significantly from those in the Health group.

Discussion
Relationship between semen HIF-1α and sperm autophagy in human In this study, we investigated the relationship between semen HIF-1α and sperm autophagy in humans.We found that incubating human semen with HIF-1α disruptors, such as 5000μΜ YC-1 or 100nM CoCl 2, resulted in an increase in HIF-1α level and sperm autophagy rate, as well as a decrease in progressive sperm motility.Previous studies have reported that patients with asthenozoospermia or varicocele exhibited significantly lower sperm motility and elevated HIF-1α levels 22,23 .Furthermore, both mouse experiment data and human clinical data have shown that sperm motility and density are significantly reduced under hypoxic conditions compared to normoxic conditions [24][25][26] .It has been observed that hypoxia-induced overexpression of HIF-1α triggers the activation of autophagy pathway 27,28 , while knockout of HIF-1α inhibits the expression of mitochondrial autophagy-related genes 29 .In this study, when human semen was incubated with 5000 µM YC-1 or 100 nM CoCl 2 for 60 min, we observed increased levels of LC3B in both YC-1 + health group and CoCl 2 + astheno group, indicating a significant increase in sperm autophagy.The energy required for sperm motility primarily comes from glycolysis and gluconeogenesis.In certain cell types, such as the granulosa cells of mice, HIF-1α-dependent autophagy has been shown to play a protective role in facilitating the switch to glycolysis, which is important for energy supply and cell survival 30 .Improving the function of sperm mitochondrial oxidative respiratory chain could increase ATP levels and significantly enhance the rate of forward-moving sperm [31][32][33] .Inhibition of glycolysis in rats has been found to dramatically reduce sperm motility and fertility 34 , and this reduction is closely correlated with HIF-1α levels 35 .In the pathway analysis conducted in this study, the differentially expressed proteins in the YC-1 + health group or CoCl 2 + astheno group were found to be enriched in various pathways, including the HIF-1 signaling pathway, glycolysis/gluconeogenesis signaling pathway, amino acid biosynthesis, and other metabolic processes.The reduction in sperm motility observed in both groups suggests that hypoxia regulates sperm autophagy and motility through these signaling pathways.This finding is consistent with previous research that highlighted the pronounced involvement of the hypoxia pathway in asthenozoospermia 16 .
YC-1 is an inhibitor of HIF-1α and is known to block the expression of HIF-1α under hypoxia conditions, thereby inhibiting its transcriptional activity.However, in this study, we observed that YC-1 did not reduce the level of HIF-1α.On the contrary, HIF-1α levels increased in the YC-1 + health group.This unexpected result may be attributed to YC-1 increasing the stability of HIF-1α rather than inhibiting its expression 36 .Consequently, HIF-1α accumulated to high level, leading to an increase of the autophagy rate.

Differentially expressed autophagy-related proteins in asthenozoospermia
The expression of various proteins is closely linked to the metabolic processes and physiological mechanisms within organisms.In this particular study, we observed distinct expression patterns in the semen samples of the YC-1 + health group and the CoCl 2 + astheno group.Specifically, we found that the protein Q0VAS5 (Histone H4) was upregulated, while the protein A0A7I2V5M3 (Cathepsin L), E9PKT6, P58499, and Q53YD7 were downregulated in the semen samples of YC-1 + health group.Similarly, the protein A0A2R8Y430 (Glutathione synthetase), Q9UM22, Q9Y6R7, P58499, and Q13438 were downregulated in the CoCl 2 + astheno group.These observed changes in protein expression align with the alterations typically observed in cases of asthenozoospermia.
Histone H4 is a critical components of nucleosomes, and modifications such as acetylation or methylation of histone H4 have been shown to impact sperm morphology and motility 37 .Studies have demonstrated that impaired exchange of histones with protamine can reduce fertilization 38 .

Table 1 .
General and clinical data of the subjects.Astheno is Asthenozoospermia.Values are mean ± SD or case number (percentage).a Analysis of variance (ANOVA) has been used.b Chi-square test has been used.c Rank sum test has been used.

Figure 3 .
Figure 3. Heat-map of differentially abundant proteins of human semen groups.

Figure 4 .
Figure 4. Protein-protein interactions (PPIs) of differentially abundant proteins in human semen after STRING analysis on YC-1 + health group (A) and in CoCl 2 + astheno group (B).

Table 2 .
Summary of the differentially expressed proteins.Astheno is Asthenozoospermia.Values are number of protein.Semen of YC-1 + health group was incubated with 5000 μM YC-1 at 37 ℃ for 60 min.Semen of CoCl 2 + astheno group was incubated with 100 nM CoCl 2 at 37 ℃ for 60 min.

Table 3 .
Effects of HIF-1α interfernts on levels of related proteins in human semen.Values are mean ± SD. Analysis of variance (ANOVA) has been used.Compared with the Health group, + p < 0.05, ++ p < 0.01; Compared with the Astheno group, **p < 0.01. HIF-